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Abstract:

Provided are topical delivery systems and methods for treating and/or
preventing a disease or disorder in a subject, or for eliciting an immune
response in a subject, including applying, to at least a portion of the
subject's post-auricular region, a topical delivery system including a
therapeutically effective amount of a topical pharmaceutical composition
comprising at least one active agent and a pharmaceutically acceptable
carrier and/or adjuvant. The topical pharmaceutical composition can
include a topical immunogenic composition. The topical delivery system
can further include a flexible substrate in communication with the
topical immunogenic composition. The subject can be a pediatric subject.

Claims:

1. A method for treating and/or preventing a disease or disorder in a
subject, comprising: applying, to at least a portion of the subject's
post-auricular region, a topical delivery system comprising a
therapeutically effective amount of a topical pharmaceutical composition
comprising at least one active agent and a pharmaceutically acceptable
carrier and/or adjuvant.

2. The method according to claim 1, wherein the disease or disorder is an
influenzae infection.

3. The method according to claim 2, wherein the influenzae infection is
selected from the group consisting of and nontypeable H. influenzae
(NTHi) infection, an H. influenzae a infection, an H. influenzae b
infection, an H. influenzae c infection, an H. influenzae d infection,
and an H. influenzae e infection.

4. The method according to claim 3, wherein the influenzae infection is a
NTHi infection.

5. The method according to claim 5, wherein the NTHi infection in a
middle ear NTHi infection, a nasopharynx NTHi infection or a lower airway
NTHi infection.

7. The method according to claim 1, wherein the at least on active agent
is an immunogenic active agent.

8. The method according to claim 6, wherein the immunogenic active agent
is at least one vaccine.

9. The method according to claim 7, wherein the immunogenic active agent
comprises a chimeric protein comprising an immunogenic PilA peptide
selected from the group consisting of the amino acid sequence of SEQ ID
NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and residues 40 to 149 of SEQ ID NO: 2,
and a portion of the LB1 peptide selected from the group consisting of
the amino acid sequence of SEQ ID NO: 4 and SEQ ID NO: 5, wherein the LB1
peptide is inserted before the immunogenic PilA peptide.

12. The method according to claim 1, wherein the topical pharmaceutical
composition comprises at least one adjuvant comprising a mucosal
adjuvant.

13. The method according to claim 12, wherein the mucosal adjuvant is
dmLT.

14. The method according to claim 13, wherein the topical delivery system
comprises a flexible substrate.

15. The method according to claim 14, wherein the flexible substrate
comprises a skin-contacting surface.

16. The method according to claim 14, wherein the flexible substrate
comprises the topical pharmaceutical composition.

17. The method according to claim 15, wherein the topical pharmaceutical
composition is provided on the skin-contacting surface.

18. The method according to claim 14, wherein the flexible substrate
comprises an backing having a lower surface and at least one active agent
containing layer comprising the at least one active agent or the topical
pharmaceutical composition, the active agent containing layer having a
skin-contacting surface provided on the lower surface of the backing.

19. The method according to claim 1, wherein applying comprises applying
the topical delivery system once a week, at weekly intervals, for a
pre-determined period of time.

20. The method according to claim 18, wherein the at least one active
agent containing layer comprises a polymeric matrix layer.

21. The method according to claim 20, wherein the active agent is
dispersed or suspended in the polymeric matrix layer.

22. The method according to claim 20, wherein the polymeric matrix layer
is a pressure-sensitive adhesive matrix layer.

23. The method according to claim 20, wherein the polymeric matrix layer
releases the active agent at a predetermined controlled rate.

24. The method according to claim 18, wherein the backing is an adhesive
backing or an occlusive adhesive backing.

25. The method according to claim 18, wherein the backing is an occlusive
backing.

26. The method of claim 1, wherein the topical delivery system does not
contain a permeation enhancer or is not applied using a penetration
enhancer.

27. The method according to claim 1, wherein applying comprises applying
the topical delivery system to the skin of the subject at a location
proximate to a lymph node in the post-auricular region of the subject.

28. The method according to claim 1, wherein the post-auricular region
does not comprise any portion of the patient's pinnae.

29. The method according to claim 1, wherein the subject is a pediatric
subject.

32. The topical delivery system according to claim 31, wherein the
topical immunogenic composition further comprises at least one
pharmaceutically acceptable topical carrier and/or adjuvant.

33. The topical delivery system according to claim 31, wherein the active
agent comprises the amino acid sequence of SEQ ID NO: 57.

34. The topical delivery system according to claim 31, wherein the
topical immunogenic composition comprises at least one adjuvant
comprising a mucosal adjuvant.

35. The topical delivery system according to claim 34, wherein the
mucosal adjuvant is dmLT.

36. The topical delivery system according to claim 31, wherein the
flexible substrate comprises a skin-contacting surface.

37. The topical delivery system according to claim 36, wherein the
flexible substrate comprises the topical pharmaceutical composition.

38. The topical delivery system according to claim 36, wherein the
topical immunogenic composition is provided on the skin-contacting
surface.

39. The topical delivery system according to claim 31, wherein the
flexible substrate comprises an backing having a lower surface and at
least one active agent containing layer comprising the at least one
active agent or the topical immunogenic composition, the active agent
containing layer having a skin-contacting surface, the active agent
containing layer provided on the lower surface of the backing such that
during use the skin-contacting surface is in contact with a skin surface
of a subject.

40. The topical delivery system according to claim 39, wherein the at
least one active agent containing layer comprises a polymeric matrix
layer.

41. The topical delivery system according to claim 40, wherein the active
agent or the topical immunogenic composition is dispersed or suspended in
the polymeric matrix layer.

42. The topical delivery system according to claim 40, wherein the
polymeric matrix layer is a pressure-sensitive adhesive matrix layer.

43. The topical delivery system according to claim 42, wherein the
wherein the active agent or the topical immunogenic composition is
dispersed or suspended in the pressure-sensitive adhesive matrix layer.

44. The topical delivery system according to claim 40, wherein the
polymeric matrix layer releases the active agent at a predetermined
controlled rate.

45. The topical delivery system according to claim 40, wherein the
polymeric matrix layer comprises one or more biodegradable or bioerodable
polymers.

46. The topical delivery system according to claim 39, wherein the
backing is an adhesive backing or an occlusive adhesive backing.

47. The topical delivery system according to claim 39, wherein the
backing is an occlusive backing.

48. The topical delivery system according to claim, wherein the topical
delivery system does not contain a permeation enhancer or is not applied
using a penetration enhancer.

49. The topical delivery system according to claim 31, wherein the
topical delivery system is configured for application to the skin of the
subject at a location proximate to a lymph node in the post-auricular
region of the subject.

50. The topical delivery system according to claim 49, wherein the
post-auricular region does not comprise any portion of the subject's
pinnae.

51. The method according to claim 50, wherein the subject is a pediatric
subject.

52. The topical delivery system according to claim 39, wherein the
topical immunogenic composition comprises at least one adjuvant
comprising a mucosal adjuvant.

53. The topical delivery system according to claim 52, wherein the
mucosal adjuvant is dmLT.

54. A method for eliciting an immune response to NTHi bacteria, in a
subject, comprising: topically administering to at least a portion of the
subject's post-auricular region, where the patient at risk of an NTHi
bacterial infection, a topical delivery system comprising a topical
immunogenic composition comprising at least one active agent, the at
least one active agent comprising an immunogenic dose of a chimeric
protein comprising an immunogenic PilA peptide selected from the group
consisting of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ
ID NO: 8, and residues 40 to 149 of SEQ ID NO: 2, and a portion of the
LB1 peptide selected from the group consisting of the amino acid sequence
of SEQ ID NO: 4 and SEQ ID NO: 5, wherein the LB1 peptide is inserted
before the immunogenic PilA peptide.

57. The method according to claim 54, wherein the topical immunogenic
composition comprises at least one mucosal adjuvant.

58. The method according to claim 57, wherein the mucosal adjuvant is
dmLT.

58. The method according to claim 54, wherein the topical delivery system
further comprises a flexible substrate.

59. The method according to claim 58, wherein the flexible substrate
comprises a skin-contacting surface.

60. The method according to claim 54, wherein the flexible substrate
comprises the topical immunogenic composition.

61. The method according to claim 59, wherein the topical immunogenic
composition is provided on the skin-contacting surface.

62. The method according to claim 58, wherein the flexible substrate
comprises an backing having a lower surface and at least one active agent
containing layer comprising at least one active agent or the topical
immunogenic composition, the active agent containing layer having a
skin-contacting surface and is provided on the lower surface of the
backing.

63. The method according to claim 62, wherein the at least one active
agent containing layer comprises a polymeric matrix layer.

64. The method according to claim 63, wherein the active agent or topical
immunogenic composition is dispersed or suspended in the polymeric matrix
layer.

65. The method according to claim 63, wherein the polymeric matrix layer
is a pressure-sensitive adhesive matrix layer.

66. The method according to claim 63, wherein the polymeric matrix layer
releases the active agent at a predetermined controlled rate.

67. The method according to claim 62, wherein the backing is an adhesive
backing or an occlusive adhesive backing.

68. The method of claim 54, wherein the topical delivery system does not
contain a permeation enhancer or is not applied using a penetration
enhancer.

69. The method according to claim 54, wherein applying to the
post-auricular region does not comprise applying to any portion of the
subject's pinnae.

70. The method according to claim 54, wherein the subject is a pediatric
subject.

71. The method according to 54, wherein administering comprises applying
the topical delivery system at least once.

72. The method according to claim 54, wherein administering comprises
applying the topical delivery system once a week, at weekly intervals,
for a pre-determined period of time.

73. The method according to claim 72, wherein the predetermined period of
time is from 2 weeks to 10 weeks.

74. The method according to claim 73, wherein the predetermined period of
time is 2 weeks.

75. The method according to claim 54, wherein the topical delivery system
remains on the patient for a period of at least 24 hours.

76. The method according to claim 54, wherein the NTHi infection is in
the middle ear, nasopharynx, or lower airway.

78. A topical delivery system for use in the treatment and/or prevention
of an influenzae infection in a subject or for use in eliciting an immune
response in a subject, comprising: a topical immunogenic composition
comprising at least one active agent comprising a chimeric protein
comprising residues 40-149 of SEQ ID NO: 2 and SEQ ID NO: 4, SEQ ID NO: 4
being inserted before residue 40 of SEQ ID NO: 2.

79. The topical delivery system according to claim 78, wherein the
influenzae infection is selected from the group consisting of and
nontypeable H. influenzae (NTHi) infection, an H. influenzae a infection,
an H. influenzae b infection, an H. influenzae c infection, an H.
influenzae d infection, and an H. influenzae e infection.

80. The topical delivery system according to claim 79, wherein the
influenzae infection is a NTHi infection.

81. The topical delivery system according to claim 80, wherein the NTHi
infection in a middle ear NTHi infection, a nasopharynx NTHi infection or
a lower airway NTHi infection.

82. The topical delivery system according to claim 78, wherein the
chimeric protein comprises the amino acid sequence of SEQ ID NO: 57.

83. The topical delivery system according to claim 78, wherein the
topical immunogenic composition comprises at least one adjuvant
comprising a mucosal adjuvant.

84. The topical delivery system according to claim 83, wherein the
mucosal adjuvant is dmLT.

85. The topical delivery system according to claim 83, wherein the
topical delivery system further comprises a flexible substrate in
communication with the topical immunogenic composition.

Description:

FIELD

[0001] The present subject matter is related to topical dosage forms and
the topical delivery of active ingredients by the administration of a
transcutaneous dosage form to the post-auricular region of a subject.

BACKGROUND

[0002] Topical dosage forms, including, e.g., creams, gels, liniments,
balms, lotions, ointments, and patches, etc., are known to be effective
delivery systems for a wide range of active ingredients. However,
formulating topical dosage forms can be difficult as challenges,
including, topical delivery specific issues, such as, penetration of the
stratum corneum, poor storage-stability and generally low patient
compliance, among others, are often not easy to overcome. Accordingly,
there is a need for an improved storage-stable, topical dosage form,
and/or method for administering the same, for treating and/or preventing
one or more disease or disorder.

SUMMARY

[0003] The present subject matter provides topical delivery systems and
methods of administering or using the same to treat or prevent diseases
and/or elicit an immune response.

[0004] The topical delivery systems may include an active ingredient. The
active ingredient may be a topical immunogenic composition. The topical
immunogenic composition may be any topical immunogenic composition,
including, e.g., a chimeric protein. The chimeric protein may include one
or more one active agents. The active chimeric protein may include
residues 40-149 of SEQ ID NO: 2 and SEQ ID NO: 4, SEQ ID NO: 4 being
inserted before residue 40 of SEQ ID NO: 2. The active chimeric protein
may include the amino acid sequence of SEQ ID NO: 57.

[0005] The topical delivery systems may include, e.g., a topical
immunogenic composition as described herein and a flexible substrate in
communication with the topical immunogenic composition. The topical
immunogenic composition may also include one or more pharmaceutically
acceptable topical carriers and/or adjuvants. The adjuvant may be any
adjuvant. For example, the adjuvant may be any known adjuvant, including
a mucosal adjuvant dmLT.

[0006] The active agent in the topical composition may be any active
agent. For example the active agent may be an immunogenic active agent.
The immunogenic active agent may be at least one vaccine or other active
agent capable of eliciting an immune response. For example, the
immunogenic active agent may include one or more chimeric protein, or
fragments thereof. In this regard the immunogenic active agent may
include an immunogenic PilA peptide selected from the group consisting of
the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and
residues 40 to 149 of SEQ ID NO: 2. The immunogenic active agent may also
include a portion of the LB1 peptide. The portion of the LB1 peptide, for
example, may be selected from the group consisting of the amino acid
sequence of SEQ ID NO: 4 and SEQ ID NO: 5, wherein the LB1 peptide is
inserted before the immunogenic PilA peptide. The PilA peptide may be any
PilA peptide. For example the PilA peptide may include, among others,
residues 40-149 of SEQ ID NO: 2 and wherein the LB1 peptide comprises SEQ
ID NO: 4. The chimeric protein comprises may include any amino acid
sequence, including, e.g., the amino acid sequence of SEQ ID NO: 57.

[0007] The topical composition may also include one or more adjuvant or
other pharmaceutically acceptable carriers. For example, the composition
may include one or more mucosal adjuvant. The mucosal adjuvant may be any
mucosal adjuvant, including, e.g., dmLT.

[0008] The topical delivery system comprises a flexible substrate. The
flexible substrate may be any flexible substrate including, e.g., a
patch, a poultice, a tape, etc. The flexible substrate may include a
skin-contacting surface. In addition, the topical the topical
pharmaceutical composition may be provided in or on the flexible
substrate.

[0009] The flexible substrate may also include a backing. The backing may
be an adhesive or occlusive backing. The backing may have one or more
surfaces upon which one or more active agent may be in direct or indirect
communication. For example, the backing may be in communication with an
active containing layer that includes the at least one active agent or
the topical pharmaceutical composition. The active agent containing layer
may have a skin-contacting surface provided on one or more surface of the
backing. The backing may include one or more polymers. The polymers may
form a matrix as a layer in communication with the backing. In addition
the polymers alone, or in combination may be adhesive. For example, a
polymer matrix layer that does or does not comprises the topical
composition may form a pressure-sensitive adhesive matrix layer. The
polymeric matrix layer may release the active agent at a predetermined
controlled rate.

[0010] According to the present methods and delivery systems, the topical
delivery system may or may not contain a permeation enhancer and/or may
or may not be applied using a penetration enhancer. Accordingly,
hydration of the skin, as well as other permeation and penetration
enhancers, are not required prior to the application of the topical
delivery system.

[0011] The topical delivery system may be applied to the skin of a subject
at a location proximate to a lymph node in the post-auricular region of
the subject. The post-auricular region according to the present subject
matter may not include of the subject's pinnae.

[0012] The present subject matter also provides methods for treating
and/or preventing a disease or disorder in a subject. The methods may
include applying, to at least a portion of the subject's post-auricular
region, a topical delivery system comprising a therapeutically effective
amount of a topical pharmaceutical composition comprising at least one
active agent and a pharmaceutically acceptable carrier and/or adjuvant.

[0013] The disease or disorder to be prevented or treated may be any known
disease disorder or pathological condition. For example, the disease or
disorder may be an influenzae infection. The influenzae infection may be
one or more of nontypeable H. influenzae (NTHi) infection, an H.
influenzae a infection, an H. influenzae b infection, an H. influenzae c
infection, an H. influenzae d infection, and/or an H. influenzae e
infection.

[0014] In the event that the disease or disorder is an NTHi infection, the
NTHi infection may be one or more of a middle ear NTHi infection, a
nasopharynx NTHi infection or a lower airway NTHi infection.

[0016] The present subject matter further provides a method for eliciting
an immune response to any bacteria. For example the bacteria may be an
NTHi bacteria, including, e.g., an H. influenzae bacteria. The bacteria
may also be, e.g, a Streptococcus pneumnoniae and/or Moraxella
catarrhalis, among others. The method may include topically administering
to at least a portion of a subject's post-auricular region, where the
patient at risk of an NTHi bacterial infection, a topical delivery system
including a topical immunogenic composition. The immunogenic composition
may include at least one active agent. The at least one active agent may
include an immunogenic dose of a protein. For example the protein may me
a chimeric protein including, e.g., an immunogenic PilA peptide selected
from the group consisting of the amino acid sequence of SEQ ID NO: 6, SEQ
ID NO: 7, SEQ ID NO: 8, and residues 40 to 149 of SEQ ID NO: 2, and a
portion of the LB1 peptide selected from the group consisting of the
amino acid sequence of SEQ ID NO: 4 and SEQ ID NO: 5. The LB1 peptide may
be inserted before the immunogenic PilA peptide. The PilA peptide may
include residues 40-149 of SEQ ID NO: 2. The LB1 peptide may include SEQ
ID NO: 4. Also, the protein may be a chimeric protein including the amino
acid sequence of SEQ ID NO: 57. The topical immunogenic composition may
also include one or more adjuvant, e.g., a mucosal adjuvant such as dmLT.

[0018] The presently described subject matter is directed to a method
wherein administering comprises applying the topical delivery system at
least once.

[0019] The presently described subject matter is directed to a method
wherein administering comprises applying the topical delivery system once
a week, at weekly intervals, for a pre-determined period of time.

[0020] The presently described subject matter is directed to a method
wherein the predetermined period of time is from 2 weeks to 10 weeks.

[0021] The presently described subject matter is directed to a method
wherein the predetermined period of time is 2 weeks.

[0022] The presently described subject matter is directed to a method
wherein the topical delivery system remains on the patient for a period
of at least 24 hours.

BRIEF DESCRIPTION OF THE FIGURES

[0023] FIG. 1 is a graphical representation of resolution of
clinically-relevant signs of experimental OM as determined by video
otoscopy and tympanometry. Boxes around days 0 and 7 on x-axis indicate
days of vaccination. *p<0.05 compared to receipt of saline;
+p<0.05 compared to receipt of dmLT alone. N=4 middle ears per
cohort on day 0; N=6 middle ears per cohort on days 3-14.

[0024] FIG. 2. is a graphical representation of eradication of from middle
ear fluids. NTHi was inoculated into the middle ears of all chinchillas
four days prior to primary immunization (inoc). Mean CFU per middle ear
fluid±SEM for each cohort is presented. Boxes around days 0 and 7 on
x-axis indicate days of vaccination. *p<0.05 compared to receipt of
saline; +p<0.05 compared to receipt of dmLT alone. N=4 middle
ears per cohort on day 0; N=6 middle ears per cohort on days 3-14.

[0025] FIG. 3 is a graphical representation of resolution of established
mucosal biofilms from within the middle ear. Mean mucosal biofilm
scores±SEM for each cohort were based on a 0-4+ scale of relative
residual mucosal biofilm. Boxes around days 0 and 7 on x-axis indicate
days of vaccination. *p<0.01 compared to receipt of saline; +p<0.01
compared to receipt of dmLT alone. N=4 middle ears per cohort on day 0;
N=6 middle ears per cohort on days 3-14.

[0026] FIG. 4 is a graphical representation of relative concentration of
adherent to middle ear mucosa. Mean CFU±SEM within biofilm that
remained adherent to the middle ear mucosa over time is presented. Boxes
around days 0 and 7 on x-axis indicate days of vaccination. *p<0.05
compared to receipt of saline; +p<0.05 compared to receipt of
dmLT alone. N=4 middle ears per cohort on day 0; N=6 middle ears per
cohort on days 3-14.

[0027] FIG. 5 is a graphical representation of secretion of cytokines and
chemokines by dermal DCs activated by TCI. Supernatants collected from
cultured chinchilla pinnae from which dermal DCs had emigrated were
assessed for the production of inflammatory mediators by membrane array.
The fold change in pixel intensity as determined by densitometry relative
to the saline-treated pinnae is shown. N=6 pinnae for each cohort.

[0028] FIG. 6 is a graphical representation of induction of polyfunctional
CD4+ T-cells within the chinchilla NALT after TCI. One week after
receipt of the second immunizing dose, the NALT from each chinchilla was
collected and processed to examine intracellular cytokine production by
CD4+ T-cells in order to discern Th phenotype. The shaded region in
each density plot indicates the positive staining for IFN-γ- and
IL-17-producing CD4+ T-cells, relative to isotype antibody controls.
One of three representative plots is presented for each cohort.

DETAILED DESCRIPTION

Definitions

[0029] The term "about" as used herein refers to a quantity, level, value,
dimension, size, or amount that varies to some extent based on the
context in which it is used. For example, such variation can be by as
much as 5%. At the least, each numerical parameter can be construed in
light of the number of reported significant digits and by applying
ordinary rounding techniques.

[0030] As used herein, the terms "administer," "administering," and
"administration," refer to any method which, in sound medical practice,
delivers the composition to a subject in such a manner as to provide a
therapeutic effect.

[0031] As used herein, the term "adjuvant" refers to a substance added to
a topical pharmaceutical composition to assist the action of the active
agent.

[0032] As used herein, the term "mucosal adjuvant" refers to protein or
oligonucleotides with immunopotentiating properties that can be
co-administered with pathogen derived antigens. Such adjuvants can
include dmLT and mutants of heat labile enterotoxin from Escherichia coli
and cholera toxin from Vibrio cholera including two mutants of the
enzymatic A subunit, LTK63 and LTR72, maintain a high degree of
adjuvanticity. LTK63 results from the substitution of serine 63 with a
lysine in the A subunit, which renders it enzymatically inactive and
non-toxic. LTR72 is derived from a substitution of alanine 72 with an
arginine in the A subunit, and has approximately 0.6% of the enzymatic
activity of wild-type LT. LTR72 is shown to be 100,000 times less toxic
than wild-type LT in Y1 cells in vitro and 25-100 times less toxic than
wild-type LT in the rabbit ileal loop assay.

[0033] As used herein, the term "particulate mucosal adjuvant" refers to
adjuvants including emulsions, microparticles, iscoms, and liposomes that
can have comparable dimensions to the pathogens that the immune system
has evolved to combat. Such particulate adjuvants can include, for
example, particles including polymers or proteins which self-assemble
into particles, biodegradable and biocompatible polyesters, the
polylactide-coglycolides (PLG), where the antigen is associated with the
particles, for example, by encapsulation or adsorption onto the particle
surface. The presently described topical pharmaceutical compositions can
comprise one or more adjuvants including one or more mucosal adjuvants
and/or particulate mucosal adjuvants.

[0034] As used herein, the term "backing" refers to a layer for
application to an area of skin that conforms to the skin.

[0035] As used herein, the term "adhesive backing" refers to a layer for
application to an area of skin of which at least a portion of the backing
adheres and conforms to the skin to which it is applied.

[0036] As used herein, the term "occlusive backing" refers to a layer for
application to an area of skin that retains moisture and heat while
increasing the concentration and absorption of an active agent being
topically applied. An occlusive backing can be an adhesive occlusive
backing. The occlusive backing conforms to the area of skin to which it
is applied.

[0037] As used herein, the phrases an "effective amount" or a
"therapeutically effective amount" of an active agent or ingredient, or
pharmaceutically active agent or ingredient, which are synonymous herein,
refer to an amount of the pharmaceutically active agent sufficient enough
to have a therapeutic effect upon administration. A therapeutically
effective amount of the pharmaceutically active agent may, will, or is
expected to cause a relief of symptoms. Effective amounts of the
pharmaceutically active agent will vary with the particular condition or
conditions being treated, the severity of the condition, the duration of
the treatment, the specific components of the composition being used, and
like factors.

[0038] The term "flexible substrate," as used herein, refers to a
substrate comprising single or multiple layers for the transdermal
delivery of one or more active agents when applied to an area of skin of
a patient, for example, a pediatric patient. Suitable flexible substrates
can include, but are not limited to, bandages, cotton pads, gauze pads,
poultices, tapes, and skin patches. Skin patches can include, but are not
limited to, "reservoir" patches and "matrix" patches. Patches may be
single- or multi-layered. A "reservoir" patch can have a liquid or gel
compartment containing the drug solution or suspension separated by a
membrane and a layer of adhesive. In a "matrix" patch, the drug can be
present, for example, dispersed or suspended, in a semi-solid or solid
layer, which may or may not be adhesive or comprise an adhesive material.
Such flexible substrates can include a backing and/or an active
containing layer. A topical pharmaceutical composition containing at
least one active agent or at least one active agent can be in contact or
communication with the backing and/or any other layer, for example a
polymeric matrix layer, provided the active agent is in contact with an
area of skin to which the system is applied, during use. One or more
active agents can be dispersed in, suspended in, or coated on a
skin-contacting surface of an active agent containing layer.

[0039] As used herein, the term "influenzae infection" refers to a
bacterial infection caused by the microbe Haemophilus influenza.

[0040] As used herein, the terms "optional" or "optionally" mean that the
subsequently described event or circumstance may or may not occur, and
that the description includes instances where said event or circumstance
occurs and instances where it does not.

[0042] As used herein, the term "polymeric matrix layer" refers to a layer
comprising one or more polymers for the release of one or more active
agents. The polymeric matrix layer can be an adhesive, i.e., an adhesive
polymeric matrix layer that adheres and conforms to an area of skin to
which it is applied. The polymeric matrix layer can be a
pressure-sensitive adhesive polymeric matrix layer, comprising for
example, one or more of polyurethane, PMMA, styrene-butadiene copolymer,
and silicone. The polymeric matrix layer can comprise one or more active
agents.

[0043] The polymer matrix layer can include an active agent homogeneously
combined in a biocompatible pressure sensitive polymer adhesive which may
or may not also contain other components. The topical delivery system can
comprise an adhesive patch having an impermeable film backing and, before
transdermal application, a release liner on the surface of the adhesive
opposite the film backing. The topical delivery system can comprise a
unit dosage form of an active agent in a topical pharmaceutical
composition that is in a matrix comprising an adhesive polymer. The
pressure sensitive adhesive and the topical pharmaceutical composition
containing the active agent can be homogeneously combined.

[0044] As used herein, the term "postauricular region" refers to the area
behind the auricle of the ear. The postauricular region may include the
skin near or proximate the postauricular lymph node. The postauricular
region does not include any portion of the pinnae.

[0045] As used herein the term "preservative" refers to any known
pharmaceutically acceptable preservative that functions by inhibiting
bacteria and/or fungi, and/or yeast, and/or mold, and/or other microbe,
and/or by inhibiting oxidation. Suitable preservatives can include but
are not limited to antimicrobial agents and/or antioxidants. Suitable
antimicrobial agents can include but are not limited to benzoates, benzyl
alcohol, sodium benzoate, sorbates, propionates, and nitrites. Suitable
antioxidants can include but are not limited to vitamin C, butylated
hydroxytoluene (BHT), sulphites, and vitamin E.

[0046] The term "prevent," "preventing," or "prevention," as used herein
refers to any reduction, no matter how slight, of a subject's
predisposition or risk for developing a disease or disorder. The term
"prevention" includes either preventing the onset of a clinically evident
disease or disorder altogether or preventing the onset of a
pre-clinically evident infection in individuals at risk. This includes
prophylactic treatment of subjects at risk of developing a disease or
disorder as presently described.

[0047] The phrase "substantially pure" as used herein refers to an
individual compound form, which is substantially devoid of all other
forms, as well as degradation products of a form, and any residual
solvent, and is at least 85 wt % pure, unless otherwise specified. The
compound form can have at least 90 wt % purity, at least 93 wt % purity,
at least 95 wt % purity, or at least 97 wt % purity.

[0048] As used herein, "subject" or "individual" or "animal" or "patient"
or "mammal," refers to any subject, particularly a mammalian subject, for
whom diagnosis, prognosis, or therapy is desired, for example, a human,
for example a pediatric human patient.

[0049] As used herein, the term "topical delivery system" refers to a
pharmaceutical dosage form that comprises a flexible substrate and at
least one topical pharmaceutical composition. The flexible substrate may
include a backing or backing layer that provides a protective outer
surface for the system, as well as a release liner or layer that covers
an adhesive portion of the system that is used to affix the same to the
skin of a patient. The release liner is removed prior to application,
thereby exposing the adhesive portion of the system, which can be a
pressure-sensitive adhesive. The topical delivery system can be
storage-stable at room temperature.

[0050] The flexible substrate can include single or multiple layers for
the transdermal delivery of one or more active agents when applied to an
area of skin of a patient, for example, a pediatric patient. Suitable
topical delivery systems can include, but are not limited to, skin
patches, including "reservoir" patches and "matrix" patches. Patches may
be single- or multi-layered. A "reservoir" patch essentially has a liquid
or gel compartment containing the active agent in solution or suspension
separated by a membrane and a layer of adhesive. In a "matrix" patch, the
active agent can be present, for example, dispersed or suspended, in a
semi-solid or solid layer, which may or may not be adhesive or comprise
an adhesive material. Such systems can include a backing or backing layer
and/or an active containing layer. An active agent can be in contact or
communication with the backing and/or any other layer, for example a
polymeric matrix layer, provided the active agent is in contact with an
area of skin to which the system is applied, during use. One or more
active agents can be dispersed in, suspended in, or coated on a
skin-contacting surface of an active containing layer.

[0051] A patch can comprise a backing layer and a reservoir provided on a
portion of the backing layer. The reservoir can be configured for
releaseably containing at least one active agent for transdermal delivery
when applied to an area of skin of a patient, for example a pediatric
patient. The reservoir can be mounted on a portion of a lower surface the
backing layer.

[0052] The patch can comprise a skin adhesive layer disposed on at least a
portion of a lower surface of the backing layer of the patch such that
the patch can be applied or releasably applied to an area of skin of the
patient.

[0053] As used herein, the term "transdermal" means the application of one
or more active agents, to the skin. Transdermal application can result in
transdermal delivery, where the active agent is delivered across one or
more layers of the skin. Likewise, as used herein, the term
"transcutaneous" means the application of a composition and/or one or
more active agents, to the epidermis which may result in the delivery of
the one or more active agents across at least the epidermis.

[0054] As used herein, the term "transcutaneous immunization" means the
application of an immunogenic substance, e.g., a vaccine onto skin, to
induce an immune response, which may include, e.g., engaging
antigen-presenting cells present within the epidermis and dermis, the
Langerhan's cells and dermal dendritic cells respectively.

[0055] As used herein, a "treatment" or "treating" of a disease, disorder,
or condition encompasses alleviation of at least one symptom thereof, a
reduction in the severity thereof, or the delay or inhibition of the
progression thereof. Treatment need not mean that the disease, disorder,
or condition is totally cured. A useful composition herein needs only to
reduce the severity of a disease, disorder, or condition, reduce the
severity of symptoms associated therewith, provide improvement to a
patient or subject's quality of life, or delay or inhibit the onset of a
disease, disorder, or condition.

[0056] Any concentration ranges, percentage range, or ratio range recited
herein are to be understood to include concentrations, percentages or
ratios of any integer within that range and fractions thereof, such as
one tenth and one hundredth of an integer, unless otherwise indicated.

[0057] Any number range recited herein relating to any physical feature,
such as polymer subunits, size or thickness, are to be understood to
include any integer within the recited range, unless otherwise indicated.

[0058] It should be understood that the terms "a" and "an" as used above
and elsewhere herein refer to "one or more" of the enumerated components.
It will be clear to one of ordinary skill in the art that the use of the
singular includes the plural unless specifically stated otherwise.
Therefore, the terms "a," "an" and "at least one" are used
interchangeably in this application. For example, "a" thickener refers to
both one thickener or a mixture comprising two or more thickeners.

[0059] Unless otherwise indicated, all numbers expressing quantities,
percentages or proportions, and other numerical values used in the
specification and claims, are to be understood as being modified in all
instances by the term "about." Accordingly, unless indicated to the
contrary, the numerical parameters set forth in the following
specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained.

[0060] Throughout the application, descriptions of various embodiments use
"comprising" language; however, it will be understood by one of skill in
the art, that in some specific instances, an embodiment can alternatively
be described using the language "consisting essentially of" or
"consisting of."

[0061] For the purpose of clarity, any element or feature of any method or
composition described herein, can be combined with any other element or
feature of any other method or composition described herein.

[0062] Other terms as used herein are meant to be defined by their
well-known meanings in the art.

NTHi Type IV Pilus (PilA) Polynucleotides and Polypeptides

[0063] The chimeric proteins of the presently described subject matter may
comprise the full length or a portion of the major subunit of the NTHi
Type IV Pilus which is encoded by the gene pilA. The PilA protein of the
NTHi isolate 86-028NP is encoded by the nucleic acid sequence set out as
SEQ ID NO: 2, which is described in U.S. patent application Ser. No.
11/019,005, incorporated by reference herein in its entirety. Also
provided are polynucleotides encoding PilA polypeptides from NTHi
clinical isolates 1728MEE, 1729MEE, 3224A, 10548MEE, 1060MEE, 1885MEE,
1714MEE, 1236MEE, 1128MEE and 214NP. The amino acid sequences of these
Pi1A polypeptides are set out in SEQ ID NOS: 34, 36, 38, 40, 42, 44, 46,
48, 50 and 52 respectively. The possibility of alternative codon usage is
specifically contemplated in polynucleotides encoding the polypeptides.
In one embodiment, the polypeptides are respectively encoded by the
nucleotide sequences set out in SEQ ID NOS: 33, 35, 37, 39, 41, 43, 45,
47, 49 and 51.

[0064] The presently described subject matter provides for polynucleotides
that hybridize under stringent conditions to (a) the complement of the
nucleotide sequences set out in SEQ ID NOS: 1, 33, 35, 37, 39, 41, 43,
45, 47, 49 and 51; (b) a polynucleotide which is an allelic variant of
any polynucleotides recited above; (c) a polynucleotide which encodes a
species homolog of any of the proteins recited above; or (d) a
polynucleotide that encodes a polypeptide comprising a specific domain or
truncation of the polypeptides of the presently described subject matter.
PilA polynucleotides from other non-typeable H. influenzae strains and
from H. influenzae strains a, b, c, e and f are specifically
contemplated. These polynucleotides can be identified and isolated by
techniques standard in the art such as hybridization and polymerase chain
reaction using part or all of the polynucleotides of SEQ ID NOS: 1, 33,
35, 37, 39, 41, 43, 45, 47, 49 and 51 as probes or primers, respectively.

[0065] The polynucleotides of the presently described subject matter also
include nucleotide sequences that are substantially equivalent to the
polynucleotides recited above. Polynucleotides according to the presently
described subject matter can have, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%,
more typically at least 90%, 91%, 92%, 93%, or 94% and even more
typically at least 95%, 96%, 97%, 98%, or 99% sequence identity to the
NTHi polynucleotides recited above.

[0066] Included within the scope of the nucleic acid sequences of the
presently described subject matter are nucleic acid sequence fragments
that hybridize under stringent conditions to the NTHi nucleotide
sequences of SEQ ID NOS: 1, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51, or
complements thereof, which fragment is greater than about 5 nucleotides,
7 nucleotides, greater than 7 nucleotides, greater than 9 nucleotides,
and greater than 17 nucleotides. Fragments of, e.g., 15, 17, or 20
nucleotides or more that are selective for (i.e., specifically hybridize
to any one of the PilA polynucleotides of the presently described subject
matter) are suitable. These nucleic acid sequence fragments capable of
specifically hybridizing to an NTHi PilA polynucleotide of the presently
described subject matter can be used as probes to detect NTHi PilA
polynucleotides of the presently described subject matter and/or can
differentiate NTHi PilA polynucleotides of the presently described
subject matter from other bacterial genes, and are preferably based on
unique nucleotide sequences.

[0067] The term "stringent" is used herein to refer to conditions that are
commonly understood in the art as stringent. Hybridization stringency is
principally determined by temperature, ionic strength, and the
concentration of denaturing agents such as formamide. Examples of
stringent conditions for hybridization and washing are 0.015 M sodium
chloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodium
chloride, 0.0015M sodium citrate, and 50% folinamide at 42° C. See
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold
Spring Harbor Laboratory, (Cold Spring Harbor, N. Y. 1989).

[0069] Other agents may be included in the hybridization and washing
buffers for the purpose of reducing non-specific and/or background
hybridization. Examples include 0.1% bovine serum albumin, 0.1%
polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium
dodecylsulfate, NaDodSO4, (SDS), ficoll, Denhardt's solution, sonicated
salmon sperm DNA (or other non-complementary DNA), and dextran sulfate,
although other suitable agents can also be used. The concentration and
types of these additives can be changed without substantially affecting
the stringency of the hybridization conditions. Hybridization experiments
are usually carried out at pH 6.8-7.4, however, at typical ionic strength
conditions, the rate of hybridization is nearly independent of pH. See
Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Ch. 4,
IRL Press Limited (Oxford, England). Hybridization conditions can be
adjusted by one skilled in the art in order to accommodate these
variables and allow DNAs of different sequence relatedness to form
hybrids.

[0070] As noted above, suitable polynucleotides are not limited to the
specific PilA polynucleotides of SEQ ID NOS: 1, 33, 35, 37, 39, 41, 43,
45, 47, 49 and 51, but also include, for example, allelic and species
variations thereof. Allelic and species variations can be routinely
determined by comparing the sequence provided in SEQ ID NOS: 1, 33, 35,
37, 39, 41, 43, 45, 47, 49 and 51, preferably the open reading frames
therein, a representative fragment thereof, or a nucleotide sequence at
least 90% identical, preferably 95% identical, to the open reading frames
within SEQ ID NOS: 1, 33, 35, 37, 39, 41, 43, 45, 47, 49 and 51 with a
sequence from another isolate of the same species or another species.
Computer program methods to determine identity and similarity between two
sequences include, but are not limited to, the GCG program package,
including GAP (Devereux et al., Nucl. Acid. Res., 12: 387, 1984; Genetics
Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN,
and FASTA (Altschul et al., J. Mol. Biol., 215: 403-410, 1990). The
BLASTX program is publicly available from the National Center for
Biotechnology Information (NCBI) and other sources (BLAST Manual,
Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra).
The well known Smith-Waterman algorithm may also be used to determine
identity.

[0071] Polynucleotides of the presently described subject matter may be
isolated from natural sources or may be synthesized by standard chemical
techniques, e.g., the phosphotriester method described in Matteucci et
al., J Am Chem Soc., 103: 3185 (1981).

[0072] The presently described subject matter provides for chimeric
proteins comprising a portion of NTHi PilA protein. The polypeptides can
comprise the NTHi 86-028NP amino acid sequences respectively set out in
SEQ ID NO: 2. Polypeptides of the presently described subject matter may
also include PilA polypeptides set out in SEQ ID NOS: 34, 36, 38, 40, 42,
44, 46, 48, 50 and 52. The PilA polypeptides of the presently described
subject matter can be those of other non-typeable H. influenzae strains
and from H. influenzae strains a, b, c, e and f.

[0073] Polypeptides of the presently described subject matter specifically
include peptide fragments (i.e., peptides) or fragments of the PilA
polypeptide that retain one or more biological or immunogenic properties
of a full length polypeptide of the presently described subject matter.
In one embodiment, PilA peptide fragments provided by the presently
described subject matter are designated TfpQ2, TfpQ3, TfpQ4 and OLP3 and
respectively comprise amino acids 35 through 68 of SEQ ID NO: 2, amino
acids 69 through 102 of SEQ ID NO: 2, amino acids 103 through 137 of SEQ
ID NO: 2, and amino acids 21 through 35 of SEQ ID NO: 2. Another PilA
peptide fragment provided by the presently described subject matter
comprises amino acids 40 through 149 of SEQ ID NO: 2.

[0074] The presently described subject matter also provides for chimeric
proteins comprising a portion of a PilA polypeptide with one or more
conservative amino acid substitutions that do not affect the biological
and/or immunogenic activity of the PilA polypeptide. Alternatively, the
PilA polypeptides of the presently described subject matter can have
conservative amino acids substitutions which may or may not alter
biological activity. The term "conservative amino acid substitution"
refers to a substitution of a native amino acid residue with a nonnative
residue, including naturally occurring and nonnaturally occurring amino
acids, such that there is little or no effect on the polarity or charge
of the amino acid residue at that position. For example, a conservative
substitution results from the replacement of a non-polar residue in a
polypeptide with any other non-polar residue. Further, any native residue
in the polypeptide may also be substituted with alanine, according to the
methods of "alanine scanning mutagenesis." Naturally occurring amino
acids are characterized based on their side chains as follows: basic:
arginine, lysine, histidine; acidic: glutamic acid, aspartic acid;
uncharged polar: glutamine, asparagine, serine, threonine, tyrosine; and
non-polar: phenylalanine, tryptophan, cysteine, glycine, alanine, valine,
proline, methionine, leucine, norleucine, isoleucine. General rules for
amino acid substitutions are set forth in Table 1 below.

[0075] The presently described subject matter also provides for chimeric
proteins comprising a portion of a variants of the NTHi PilA polypeptides
of the presently described subject matter (e.g., a polypeptide exhibiting
at least about 65%, at least about 70%, at least about 75%, at least
about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%,
91%, 92%, 93%, 94%, at least about 95%, 96%, 97%, at least about 98%, or
at least about 99% amino acid identity to a polypeptide of SEQ ID NOS: 2,
34, 36, 38, 40, 42, 44, 46, 48, 50 and 52) that retain biological and/or
immunogenic activity.

[0076] The presently described subject matter is directed to PilA
polynucleotides of the presently described subject matter that may be
inserted in a vector for amplification or expression. For expression, the
polynucleotides are operatively linked to appropriate expression control
sequences such as promoter and polyadenylation signal sequences. Further
provided are host cells comprising polynucleotides of the presently
described subject matter. Exemplary prokaryotic host cells can include
bacteria such as E. coli, Bacillus, Streptomyces, Pseudonzonas,
Salmonella, and Serratia. The presently described subject matter is
directed to methods of producing the polypeptides of the presently
described subject matter by growing the host cells and isolating
polypeptide from the host cells or growth medium. Alternatively,
polypeptides of the presently described subject matter can be prepared by
chemical synthesis using standard means. Particularly convenient are
solid phase techniques (see, e.g., Erikson et al., The Proteins (1976) v.
2, Academic Press, New York, p. 255). Automated solid phase synthesizers
are commercially available. In addition, modifications in the sequence
are easily made by substitution, addition or omission of appropriate
residues. For example, a cysteine residue may be added at the carboxy
terminus to provide a sulfhydryl group for convenient linkage to a
carrier protein, or spacer elements, such as an additional glycine
residue, may be incorporated into the sequence between the linking amino
acid at the C-terminus and the remainder of the peptide.

[0077] The term "isolated" refers to a substance removed from, and
essentially free of, the other components of the environment in which it
naturally exists. For example, a polypeptide is separated from other
cellular proteins or a DNA is separated from other DNA flanking it in a
genome in which it naturally occurs.

[0078] Recombinant PilA protein (rPilA) may be generated to serve as a
more readily renewable product. To do this, the published protocol of
Keizer et al. (J. Biol. Chem., 276: 24186-14193, 2001), who studied a
pilin which also had four Cys residues as it will be critical that rPilA
similarly be properly folded so as to possess functional qualities of the
native pilin subunit, is utilized. Briefly, a truncated pilin is
engineered wherein the first 28 residues are removed from the N-terminus
to prevent aggregation, and this truncated pilin will be further
engineered to be transported to the periplasm by means of the
incorporation of an OmpA leader sequence in the construct. Using this
strategy Keizer et al. generated a recombinant soluble monomeric P.
aeruginosa pilin protein that was able to bind to its receptor (asialo
GM1) in in vitro assays and decrease morbidity and mortality in mice when
the peptide was delivered 15 minutes prior to heterologous challenge.
This soluble, monomeric, truncated form of NTHi PilA will be useful in
the studies described herein.

[0079] The presently described subject matter also provides for synthetic
chimeric proteins. The chimeric proteins may be synthesize, purified and
sequenced using standard techniques. For example, the chimeric proteins
may be assembled semi-manually by stepwise Fmoc-tert-butyl solid-phase
synthesis and purified by HPLC. The composition and amino acid sequence
of recombinant and synthetic chimeric proteins may be confirmed by amino
acid analysis and/or mass spectral analysis.

Antibodies

[0080] The presently described subject matter provides antibodies which
bind to antigenic epitopes of the chimeric proteins of the presently
described subject matter. The antibodies may be polyclonal antibodies,
monoclonal antibodies, antibody fragments which retain their ability to
bind their unique epitope (e.g., Fv, Fab and F(ab)2 fragments), single
chain antibodies and human or humanized antibodies. Antibodies may be
generated by techniques standard in the art using chimeric protein(s) of
the presently described subject matter or host cells expressing chimeric
protein(s) of the presently described subject matter as antigens.

[0081] The presently described subject matter provides for antibodies
specific for the chimeric proteins of the presently described subject
matter and fragments thereof, which exhibit the ability to kill both H.
influenzae bacteria and to protect humans from infection. The presently
described subject matter also provides for antibodies specific for the
chimeric proteins of the presently described subject matter which reduce
the virulence, inhibit adherence, inhibit biofilm formation, inhibit
twitching motility, inhibit cell division, and/or inhibit penetration
into the epithelium of H. influenzae bacteria and/or enhance phagocytosis
of the H. influenzae bacteria.

[0083] It is also possible to confer short-term protection to a host by
passive immunotherapy via the administration of pre-formed antibody
against a chimeric protein of the presently described subject matter.
Thus, antibodies of the presently described subject matter may be used in
passive immunotherapy. Human immunoglobulin is preferred in human
medicine because a heterologous immunoglobulin may provoke an immune
response to its foreign immunogenic components. Such passive immunization
can, for example, be used on an emergency basis for immediate protection
of unimmunized individuals subject to special risks.

[0084] Antibodies of the presently described subject matter may be used in
the production of anti-idiotypic antibody, which in turn can be used as
an antigen to stimulate an immune response against the chimeric protein
epitopes or H. influenzae epitopes.

Methods for Eliciting an Immune Response and Compositions Therefor

[0085] The presently described subject matter is directed to methods of
eliciting in an individual an immune response to a bacteria. For example,
the bacteria may be an NTHi bacteria, including, e.g., an H. influenzae
bacteria. The bacteria may also be, e.g., a Streptococcus pneumnoniae
and/or Moraxella catarrhalis, among others. H. influenzae. For example,
the methods can elicit an immune response to the chimeric proteins of the
presently described subject matter. These methods can elicit one or more
immune responses, including but not limited to, immune responses which
inhibit bacterial replication, immune responses which block H. influenzae
adherence to cells, immune responses which prevent H. influenzae
twitching, immune responses that kill H. influenzae bacteria and immune
responses which prevent biofilm formation. The methods can comprise a
step of topically administering an immunogenic dose of a composition
comprising one or more chimeric proteins of the presently described
subject matter. The methods can comprise topically administering an
immunogenic dose of a composition comprising a cell expressing one or
more chimeric proteins of the presently described subject matter.
Further, the methods can comprise topically administering an immunogenic
dose of a composition comprising one or more polynucleotides encoding one
or more chimeric proteins of the presently described subject matter. The
polynucleotide may be a naked polynucleotide not associated with any
other nucleic acid or may be in a vector such as a plasmid or viral
vector (e.g., adeno-associated virus vector or adenovirus vector). The
methods may be used in combination in a single individual. The methods
may be used prior or subsequent to H. influenzae infection of an
individual. The methods and compositions of the presently described
subject matter may be used to topically treat or prevent any pathological
condition involving H. influenzae (typeable and nontypeable strains) such
as OM, pneumonia, sinusitis, septicemia, endocarditis, epiglottitis,
septic arthritis, meningitis, postpartum and neonatal infections,
postpartum and neonatal sepsis, acute and chromic salpingitis,
epiglottis, pericardis, cellulitis, osteomyelitis, endocarditis,
cholecystitis, intraabdominal infections, urinary tract infection,
mastoiditis, aortic graft infection, conjunctitivitis, Brazilian purpuric
fever, occult bacteremia, and chronic obstructive pulmonary disease and
exacerbation of underlying lung diseases such as chronic bronchitis,
bronchietasis, and cystic fibrosis.

[0086] In methods of the presently described subject matter, a composition
of the presently described subject matter can be topically administered
as a priming dose followed by one or more topical booster doses. Proteins
or polypeptides that beneficially enhance the immune response such as
cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g.
Leaf) or co-stimulatory molecules can be topically co-administered.

[0087] An "immunogenic dose" of a composition of the presently described
subject matter is one that generates, after topical administration, a
detectable humoral (antibody) and/or cellular (T cell) immune response in
comparison to the immune response detectable before topical
administration or in comparison to a standard immune response before
topical administration. The immune response resulting from the presently
described methods may be protective and/or therapeutic. The antibody
and/or T cell immune response can protect the individual from H.
influenzae infection, for example, infection of the middle ear and/or the
nasopharynx or lower airway. In this use, the precise dose depends on the
patient's state of health and weight, topical administration, the nature
of the formulation, etc., but generally ranges from about 1.0 μg to
about 5000 μg per 70 kilogram patient, more commonly from about 10 to
about 500 μg per 70 kg of body weight.

[0088] Humoral immune response may be measured by many well known methods,
such as Single Radial Immunodiffussion Assay (SRID), Enzyme Immunoassay
(ETA) and Hemagglutination Inhibition Assay (HAI). In particular, SRID
utilizes a layer of a gel, such as agarose, containing the immunogen
being tested. A well is cut in the gel and the serum being tested is
placed in the well. Diffusion of the antibody out into the gel leads to
the formation of a precipitation ring whose area is proportional to the
concentration of the antibody in the serum being tested. ETA, also known
as ELISA (Enzyme Linked Immunoassay), is used to determine total
antibodies in the sample. The immunogen is adsorbed to the surface of a
microtiter plate. The test serum is exposed to the plate followed by an
enzyme linked immunoglobulin, such as IgG. The enzyme activity adherent
to the plate is quantified by any convenient means such as
spectrophotometry and is proportional to the concentration of antibody
directed against the immunogen present in the test sample. HAI utilizes
the capability of an immunogen such as viral proteins to agglutinate
chicken red blood cells (or the like). The assay detects neutralizing
antibodies, i.e., those antibodies able to inhibit hemagglutination.
Dilutions of the test serum are incubated with a standard concentration
of immunogen, followed by the addition of the red blood cells. The
presence of neutralizing antibodies will inhibit the agglutination of the
red blood cells by the immunogen. Tests to measure cellular immune
response include determination of delayed-type hypersensitivity or
measuring the proliferative response of lymphocytes to target immunogen.

[0089] The presently described subject matter correspondingly provides
topical compositions suitable for eliciting an immune response to
chimeric proteins of the presently described subject matter. As noted
above, the topical compositions can comprise one or more chimeric
proteins, cells expressing one or more chimeric proteins, or one or more
polynucleotides encoding one or more chimeric proteins. The topical
compositions may also comprise one or more other ingredients such as
pharmaceutically acceptable topical carriers and adjuvants.

[0090] In topical compositions of the presently described subject matter,
a chimeric protein may be fused to another protein when produced by
recombinant methods. For example, the other protein may not, by itself,
elicit antibodies, but it may stabilize the first protein and forms a
fusion protein retaining immunogenic activity. Further, the fusion
protein can comprise another protein that is immunogenic, such as
Glutathione-S-transferase (GST) or beta-galactosidase, relatively large
co-proteins which solubilize the fusion protein and facilitate production
and purification thereof. The other protein may act as an adjuvant in the
sense of providing a generalized stimulation of the immune system. The
other protein may be fused to either the amino or carboxy terminus of the
chimeric proteins of the presently described subject matter.

[0091] In other topical compositions of the presently described subject
matter, chimeric proteins may be otherwise linked to carrier substances.
Any method of creating such linkages known in the art may be used.
Linkages can be formed with hetero-bifunctional agents that generate a
disulfide link at one functional group end and a peptide link at the
other, such as a disulfide amide forming agent, e.g.,
N-succidimidyl-3-(2-pyridyldithio) proprionate (SPDP) (see, e.g., Jansen
et al., Inimun. Rev. 62:185, 1982) and bifunctional coupling agents that
form a thioether rather than a disulfide linkage such as reactive esters
of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid,
4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid and the like, and
coupling agent which activate carboxyl groups by combining them with
succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, for sodium salt such as
succinimmidyl 4-(N-maleimido-methyl) cyclohexane-1-carobxylate (SMCC).

[0092] The present chimeric proteins may be formulated as neutral or salt
forms. Pharmaceutically acceptable salts, include the acid addition salts
(formed with the free amino groups of the peptide) and which are formed
with inorganic acids such as, e.g., hydrochloric or phosphoric acids, or
such organic acids as acetic, oxalic, tartaric, mandelic. Salts formed
with the free carboxyl groups may also be derived from inorganic bases
such as, e.g., sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine, trimethylamine,
2-ethylamino ethanol, histidine, and procaine.

[0093] Topical compositions of the presently described subject matter may
further comprise one or more adjuvants, including for example, known
adjuvants. Known adjuvants include, for example, emulsions such as
Freund's Adjuvants and other oil emulsions, Bordetella pertussis, MF59,
purified saponin from Quillaja saponaria (QS21), aluminum salts such as
hydroxide, phosphate and alum, calcium phosphate, (and other metal
salts), gels such as aluminum hydroxide salts, mycobacterial products
including muramyl dipeptides, solid materials, particles such as
liposomes and virosomes. Examples of natural and bacterial products known
to be used as adjuvants include monophosphoryl lipid A (MPL), RC-529
(synthetic MPL-like acylated monosaccharide), OM-174 which is a lipid A
derivative from E. coli, holotoxins such as cholera toxin (CT) or one of
its derivatives, pertussis toxin (PT) and heat-labile toxin (LT) of E.
coli or one of its derivatives, and CpG oligonucleotides. Adjuvant
activity can be affected by a number of factors, such as carrier effect,
depot formation, altered lymphocyte recirculation, stimulation of T-25
lymphocytes, direct stimulation of B-lymphocytes and stimulation of
macrophages.

[0094] Topical compositions of the presently described subject matter can
be in any form suitable for topical delivery. Such formulations can be
topically administered in combination with a flexible substrate. The
active topically immunogenic ingredient can be encapsulated, for example,
in the form of nanocapsules or microcapsules; can be mixed with
excipients that are topically pharmaceutically acceptable and compatible
with the active ingredient. Suitable excipients are, e.g., water, saline,
dextrose, glycerol, or the like and combinations thereof. In addition, if
desired, the topical vaccine may contain minor amounts of auxiliary
substances such as wetting or emulsifying agents, pH buffering agents, or
adjuvants, which can enhance the effectiveness of the topical vaccine.
The topical vaccines are administered topically.

[0095] Topical formulations may include carriers, for example,
polyalkalene glycols or triglycerides. Topical formulations may contain
10%-95% of active ingredient, or 25-70% of active ingredient.

[0097] Topical formulations may be presented in unit-dose or multi-dose
containers.

Methods of Inhibiting H. influenzae

[0098] The presently described subject matter also includes methods of
inhibiting H. influenzae type IV pili function in an individual. The
methods comprise topically administering to the individual, for example,
one or more antibodies of the presently described subject matter and/or
one or more chimeric proteins of the presently described subject matter;
in an amount that inhibits function of the pili. In vitro assays may be
used to demonstrate the ability to inhibit pili function. These methods
can include, for example, methods using inhibitors of adherence mediated
via type IV pili, inhibitors that disrupt existing biofilms mediated by
type IV pili, and inhibitors of twitching.

[0100] Topical compositions comprising inhibitors of H. influenzae type IV
pili function are provided. The topical compositions may comprise or
consist of one of the foregoing active ingredients alone, may comprise
combinations of the foregoing active ingredients or may comprise
additional active ingredients used to treat bacterial infections. As
discussed above, the topical compositions may comprise one or more
additional ingredients such as pharmaceutically effective topical
carriers. Also as discussed above, dosage and frequency of the topical
administration of the topical compositions are determined by standard
techniques and depend, for example, on the weight and age of the
individual, area of topical administration, and the severity of symptoms.
Topical administration of the pharmaceutical compositions may be, for
example, by application to an area of skin, oral, buccal, nasal, rectal,
intranasal, or vaginal.

Kinetic Analysis and Evaluation of the Mechanisms Involved in the
Resolution of Experimental Nontypeable Haemophilus influenza
("NTHi")-Induced Otitis Media after Transcutaneous Immunization ("TCI")

[0103] The mechanisms for the observed rapid resolution of established
NTHi-induced OM after TCI with chimV4+ dmLT in a chinchilla model (of
nontypeable Haemophilus influenzae (NTHi)-induced otitis media) ("OM"))
were analyzed. The kinetics of disease resolution were assessed over a
period of 14 days via multiple parameters. Video otoscopy and
tympanometry were performed as clinically-relevant evaluations of the
incidence and severity of OM in addition to examination of mucosal
biofilms within the middle ear to rank disease severity grossly. Culture
of NTHi from middle ear fluids and mucosal biofilms provided an
assessment of the bacterial load within both the planktonic and adherent
populations, respectively, over time. The quantity of immunogen-specific
antibody in middle ear fluids was determined to identify the point at
which an adaptive immune response contributed to disease resolution.
Moreover, as it became apparent that innate immune elements within the
middle ear mucosa played a role early in this disease resolution process,
the relative amount of the host defense molecule chinchilla 3-defensin-1
(cBD-1, an orthologue of human 3-defensin 3), was examined. Lastly, the
functional activation of dermal DCs induced by TCI of chinchilla pinnae
and the phenotype of the resultant CD4+ T-cell response was analyzed.
Collectively, these data supported a proposed model wherein TCI with a
chimeric immunogen directed against two critical NTHi adhesions and
delivered with a potent adjuvant stimulated the activation and migration
of dermal DCs to the NALT, the expansion and differentiation of CD4+
T-cells to a polyfunctional phenotype and subsequent production of
specific antibody to facilitate the eradication of NTHi from the middle
ear and resolution of both established mucosal biofilms resident within
the middle ear and active disease.

[0104] A. Materials and Methods

[0105] A (i) Animals

[0106] Sixty-one adult chinchillas (Chinchilla lanigera; Rauscher's
Chinchilla Ranch, LaRue, Ohio; mean mass 550±12 g) with no evidence of
middle ear disease as determined by video otoscopy (MedRx, Largo, Fla.)
and tympanometry (EarScan, Murphy, N.C.) were enrolled and divided into 3
cohorts of 20-21 chinchillas each. Animal care and all procedures were
performed in concordance with institutional and federal guidelines, and
were conducted under an approved protocol.

[0107] A (ii) Immunogen and Adjuvant

[0108] chimV4 is a novel, chimeric immunogen wherein a modified and
N-terminally truncated form of PilA, the majority subunit of Tfp, serves
as an immunogenic carrier for 24 amino acids of a B-cell epitope from
surface-exposed region 3 of OMP P5. A double mutant form of E. coli
heat-labile enterotoxin, called LT(R192G-L211A) and abbreviated `dmLT`,
wherein glycine is substituted for arginine at position 192 and alanine
is substituted for lysine at position 211, served as the adjuvant. The
amino acid substitutions render dmLT nontoxic while maintaining adjuvant
properties.

[0109] A (iii) Bacterial Strain

[0110] NTHi strain 86-028NP was isolated from the nasopharynx of a child
undergoing tympanostomy and tube insertion for chronic OM at Nationwide
Children's Hospital, Columbus, Ohio. This strain has been characterized
and extensively used in chinchilla models of OM, a rat model of pulmonary
clearance and a murine model of OM.

[0111] A (iv) NTHi Challenge and Transcutaneous Immunization Regimen

[0112] Chinchillas were challenged with NTHi exclusively transbullarly
with 1000 CFU strain 86-028NP delivered in 0.3 ml sterile, pyrogen-free
0.9% sodium chloride (Hospira, Inc., Lake Forest, Ill.) per bulla and the
challenge dose was confirmed by plate count. This challenge model has
been shown to result in the formation of a mucosal biofilm in >83% of
middle ears within four days. Four days after challenge (day 0), all
animals were immunized by TCI. The pinnae of alert animals was first
hydrated by placement of gauze soaked in sterile, pyrogen-free 0.9%
sodium chloride on the inner surface for 5 min. Pinnae were then blotted
with dry gauze and 50 μl of each vaccine formulation was applied to
the center of the inner face of each pinna using a pipet. The pinnae were
then folded in half and opposing surfaces gently rubbed together.
Formulations consisted of 10 μg chimV4 admixed with 10 μg dmLT, 10
μg dmLT alone or 50 μl pyrogen-free 0.9% sodium chloride and were
delivered twice at a weekly interval (day 0 and day 7).

[0113] A (v) Otoscopy and Tympanometry

[0114] Video otoscopy using a 0-degree, 3-in. probe connected to a digital
camera system (MedRx, Largo, Fla.) was utilized to monitor signs of
tympanic membrane inflammation and/or presence of fluid within the middle
ear space. Middle ear pressure, tympanic membrane compliance and tympanic
width were monitored via tympanometry using a MADSEN OTOflex 100 (GN
Otometrics, Schaumburg, Ill.). Overall signs of OM were blindly rated on
a scale of 0 to 4+. Middle ears with a score of 2.0 were consistently
considered positive for otitis media as middle ear fluid (MEF) was
visible behind the tympanic membrane. Each middle ear was considered
independent (n=4 middle ears per cohort on day 0, n=6 middle ears per
cohort for days 3-14), and for each cohort, the percentage of middle ears
with OM was calculated.

[0115] A (vi) Collection of Samples

[0116] On days 0, 3, 5, 7, 9, 11 and 14 after primary TCI, three
chinchillas from each cohort were sacrificed. MEF was collected by
epitympanic tap, serially diluted and plated on to chocolate agar to
semi-quantitate CFU planktonic NTHi. Fluid from each middle ear was
considered independent (n=4 middle ears per cohort on day 0, n=6 middle
ears per cohort for days 3-14), and for each cohort, the mean CFU NTHi/ml
middle ear fluid±SEM was presented. Middle ear mucosal biofilm, if
present, was collected after evaluation as described below, homogenized
and plated on to chocolate agar to semi-quantitate the CFU NTHi adherent
to the middle ear mucosa. Each middle ear was considered independent (n=4
middle ears per cohort on day 0, n=6 middle ears per cohort for days
3-14) and for the cohort, the mean CFU NTHi/mg tissue±SEM was
presented.

[0117] A (vii) Evaluation of mucosal biofilms within the middle ear

[0118] Upon sacrifice, the inferior bullae from each animal were
dissected, opened to reveal the middle ear space and washed with 1.0 ml
sterile, pyrogen-free 0.9% sodium chloride to remove residual MEF and
loosely adherent bacterial biofilm. The bullae and remaining adherent
mucosal biofilm were then imaged with a digital camera. Each image was
blindly ranked on the relative amount of residual mucosal biofilm using a
0-4+ scale, wherein 0=no mucosal biofilm; 1=mucosal biofilm fills <25%
of middle ear space; 2=mucosal biofilm fills 25-50% of middle ear space;
3=mucosal biofilm fills 50-75% of middle ear space and 4=mucosal biofilm
fills 75-100% of middle ear space. The mean mucosal biofilm score±SEM
for the cohort (n=4 middle ears per cohort on day 0, n=6 middle ears per
cohort for days 3-14) was reported.

[0119] A (viii) Enzyme-Linked Immunosorbent Assays

[0120] To determine the relative quantity of immunogen-specific IgG and
IgA in clarified, pooled MEFs, endpoint ELISA was performed. Samples were
incubated in chimV4-coated wells (0.2 g protein/well) for 3 h at
25° C. and bound antibody was detected with HRP-conjugated goat
anti-rat IgG or IgA (Bethyl Laboratories, Montgomery, Tex.). Color was
developed with 3,3',5,5'-tetramethylbenzidine (TMB; Pierce Biotechnology,
Rockford, Ill.). Endpoint reciprocal titers were defined as the dilution
that yielded an OD450 nm value of 0.1 above control wells that were
incubated without sample fluids. Assays were performed a minimum of three
times and reciprocal titers reported as the geometric mean (GMT) with 95%
confidence intervals.

[0121] To quantitate the host defense peptide cBD-1 within mucosal
homogenates collected on day 3, a sandwich ELISA was performed. Clarified
homogenates of middle ear mucosa and mucosal biofilm were incubated in
wells of a Microfluor 2 black U-bottom microtiter plate (Thermo
Scientific, Rochester, N.Y.) coated with Protein G-purified rabbit
anti-recombinant cBD-1 (0.5 g antibody/well) overnight at 4° C.
Host defense peptide was detected by addition of FITC-conjugated purified
rabbit anti-recombinant cBD-1. The concentration of cBD-1 in each mucosal
sample was calculated by comparison of the mean fluorescence of each well
versus a standard curve generated with purified recombinant cBD-1. The
mean concentration of cBD-1±SEM for each cohort from three independent
assays is reported.

[0122] A (ix) Functional Responses of Dermal DCs after TCI

[0123] To evaluate functional responses of cutaneous DCs induced by TCI,
the efflux of dermal DCs from the chinchilla pinnae and secretion of
immune effectors was examined. Three hours after TCI with chimV4 admixed
with 10 μg dmLT, 10 examined g dmLT alone, or sterile, pyrogen-free
0.9% sodium chloride, three chinchillas per formulation were sacrificed
and both pinnae removed, placed in 70% v/v ethanol for 5 min. and
transferred to sterile petri dishes to dry. The pinnae were trimmed to
2.5 cm2, the dorsal and ventral faces peeled apart, cartilage
removed and the pinnae placed on to 8 μm pore size Transwell
membranes. Two milliliters of RPMI 1640 (Corning cellgro, Manassas, Va.)
plus 0.5% (w/v) bovine serum albumin (Sigma Aldrich) were added to the
basolateral chamber and incubated for 20 h at 37° C., 5% CO2,
in a humidified atmosphere. Cells that had migrated into the basolateral
chamber were separated based on expression of CD11c+ using MACS
magnetic microbeads (Miltenyi Biotech, Cambridge, Mass.) then incubated
with antibody directed against CD11 b (eBiosciences, Inc., San Diego,
Calif.) and DC-SIGN (R&D Systems, Minneapolis, Minn.) for discrimination
of dermal DCs. The number of dermal DC per ml culture medium was
determined as the number of events detected within a 25 μl volume
using a calibrated Accuri C6 flow cytometer (BD Biosciences, Sparks,
Md.). Each pinna was considered independent and the mean±SEM for each
cohort presented (N=6 pinnae per cohort).

[0124] The clarified supernatants from each of the six cultured pinnae per
cohort described above were applied to Proteome profiler human cytokine
array kit (R&D Systems) and assayed following the manufacturer's
instructions. Relative pixel intensity was determined with a BioRad GS800
densitometer and analyzed with Quantity One software (BioRad, Hercules,
Calif.). The fold increase in pixel intensity between pinnae collected
from chinchillas administered dmLT alone or chimV4+ dmLT was compared to
pinnae administered saline.

[0125] A (x) Determination of T-Cell Phenotype

[0126] It has been shown that dendritic cells migrate to the NALT after
TCI of the chinchilla pinnae, indicating that this lymphoid aggregate may
serve as an immune inductive site. To examine the phenotype of the immune
response induced following TCI, cytokine production by CD4+ T-cells
within the NALT was examined. One week after receipt of the second
immunizing dose as described above, the NALT was collected from three
chinchillas per cohort; homogenized individually using GentleMACS
dissociator (Miltenyi Biotech) and CD3+ cells isolated using MACS
magnetic microbeads. A total of 1×106 cells/0.5 ml RPMi 1640
plus 0.5% (w/v) bovine serum albumin was incubated with Leukocyte
activation cocktail plus GolgiPlug (BD Biosciences) for 5 h at 37°
C., 5% CO2 in a humidified atmosphere prior to stain with human
Th1/Th2/Th17 phenotyping kit according to manufacturer's instructions (BD
Biosciences). 20,000 CD4+ lymphocytes were acquired using an Accuri
C6 flow cytometer and data analyzed with FloJo software (Tree Star, Inc.,
Ashland, Oreg.). One of three representative assays is presented.

[0127] A (xi) Statistical Analyses

[0128] Data analyses were performed using GraphPad Prism v. 5.01 (La
Jolla, Calif.). Statistical differences among cohorts in bacterial
concentration within MEFs and mucosal biofilms, antibody titers and
dendritic cell migration were determined using Kruskal-Wallis one-way
analysis of variance on ranks and Dunn's method for multiple comparisons.
A p-value of ≦0.05 was considered significant. Significant
differences in the percentage of middle ears with OM and differences
among mucosal biofilm scores were analyzed by repeated measures analysis
of variance and Bonferroni's multiple comparison test. A p-value of
≦0.05 was considered significant.

[0129] B. Results

[0130] B (i) Resolution of Established Experimental Otitis Media

[0131] As a clinically-relevant assessment for the resolution of
established experimental OM, each tympanic membrane was examined to
document signs of disease including erythema, the presence of middle ear
fluid behind the tympanic membrane and changes in tympanometric read
outs. As expected, on day 0, 100% of middle ears (122/122) were positive
for the presence of MEF behind the tympanic membrane as each middle ear
had been directly inoculated with NTHi four days prior (FIG. 1). Within
three days after receipt of the first immunizing dose, a 33% (2/6 middle
ears) reduction in OM was observed after receipt of the adjuvant dmLT
alone; however, within seven days after administration of the primary
dose, the percentage of animals with OM increased to 100% (6/6 middle
ears). The reduction in the incidence of OM after receipt of dmLT showed
that an initial, non-specific immune response was induced following
administration of this potent adjuvant, a trend observed again after
receipt of the second immunizing dose on day 7. Delivery of chimV4
admixed with dmLT resulted in a greater reduction in the percentage of
middle ears with OM to 50% (3/6 middle ears) three and five days after
receipt of the first dose, statistically significant results compared to
receipt of saline (p<0.05). Moreover, seven days after primary TCI
with chimV4 admixed with dmLT only 17% (1/6 middle ears) demonstrated
signs on OM and although this percentage increased to 33% (2/6 middle
ears) on day nine, receipt of the second immunizing dose subsequently
induced rapid and complete resolution of experimental disease. On each
day after receipt of the second immunizing dose, significantly fewer
middle ears in the cohort that received chimV4+ dmLT exhibited signs of
experimental OM, compared to either cohort that received saline or dmLT
alone (p<0.05). These results indicated that incorporation of chimV4
with dmLT facilitated an enhanced and NTHi-targeted immune response.
Moreover, these data demonstrated that delivery of chimV4+ dmLT induced
an immune response in which clinically-relevant signs of OM were
abrogated by 50% within three days after administration and experimental
disease had resolved within 14 days.

[0132] B (ii) Eradication of from Middle Ear Fluids

[0133] To examine clearance of planktonic NTHi after TCI, MEFs were
collected when their presence was indicated by video otoscopy and
cultured to determine the relative concentration of bacteria within these
fluids. On day 0, four days after direct challenge of middle ears with
1000 CFU NTHi, and prior to administration of any vaccine formulation,
the inoculum had multiplied to 9.7×106 to 1.1×107
CFU/ml in all animals (FIG. 2), as is expected in this challenge model.
No significant difference in bacterial concentration was observed among
the three cohorts until one week after receipt of the primary immunizing
dose. Whereas beginning on day 3 a significant 50% reduction in middle
ears with signs of OM in the cohort that received chimV4+ dmLT was
detected by video otoscopy and tympanometry (see FIG. 1), the bacterial
concentration within the MEFs of chinchillas observed to have OM ranged
from 2.4×107 to 5.0×107 CFU/ml and did not allow
for statistical discrimination from the cohorts administered saline or
dmLT only (1.0×107 to 5.3×107 CFU/ml). However, as
shown in FIG. 2, beginning seven days after primary TCI, and specifically
upon receipt of the second immunizing dose, a rapid and significant 6-log
reduction in NTHi concentration within middle ear fluids of animals
administered chimV4+ dmLT was observed, compared to receipt of saline or
dmLT alone (p<0.05), with complete eradication of NTHi from the middle
ears of animals in this cohort by day 14. These data demonstrated that
TCI with chimV4+ dmLT induced an immune response that effectively
contributed to the elimination of planktonic NTHi within middle ear
fluids, a response that was most effective after administration of the
boosting dose.

[0134] B (iii) Resolution of Established Mucosal Biofilms from the Middle
Ear after Direct Challenge

[0135] To evaluate the kinetics of mucosal biofilm resolution afforded by
TCI, the middle ears of all chinchillas were inoculated with and a robust
biofilm allowed to form. In this model, >83% of middle ears develop a
mucosal biofilm that fills 75-100% of the middle ear space within four
days. Following immunization, any residual biofilm was evaluated and
ranked on a 0 to 4+ scale wherein a score of 0 indicated that no mucosal
biofilm was present and 4+ designated that 75-100% of the middle ear
space contained an mucosal biofilm. As shown in FIG. 3, on day 0 (four
days after direct challenge of the middle ear), the mean mucosal biofilm
scores for each of the three cohorts ranged from 3.7 to 4.0, which
indicated that 75-100% of the middle ear space of all animals contained a
mucosal biofilm, as expected for this model. No change in rank was
observed in the cohort that received saline, as a mean score of 3.0 was
maintained for the duration of the study. Receipt of dmLT alone, however,
afforded a 25% reduction in mucosal biofilm with scores of 2.3-2.8
detected after receipt of the second dose. These data provided additional
evidence to support the induction of a nonspecific immune response by
this powerful adjuvant.

[0136] Notably, administration of chimV4 with dmLT resulted in a
statistically significant reduction in mucosal biofilm from the middle
ear five days after primary TCI, compared to receipt of saline
(p<0.01). At this time point, the mean biofilm score equaled
2.3±0.2 and indicated that approximately 50% of the middle ear space
contained a mucosal biofilm. Over time, biofilm scores for this cohort
consistently decreased; seven days after primary TCI, a rank of
1.7±0.2 was achieved (25-50% of the middle ear space had a mucosal
biofilm) and by day 14, 75% of the biofilm was eliminated (p<0.01
compared to saline and dmLT cohorts). These data demonstrated that TCI
with chimV4+ dmLT was effective to stimulate the resolution of an
established mucosal biofilm from the middle ear.

[0137] B (iv) Eradication of NTHi from Established Mucosal Biofilms in the
Middle Ear

[0138] To examine the relative concentration of NTHi resident within a
biofilm adherent to the mucosa in the middle ear, compared to the
planktonic population available for culture from MEFs as described in
FIG. 2, the middle ear mucosa and accompanying NTHi biofilm was
collected, homogenized and cultured. Four days after direct inoculation
of the middle ear, the mean bacterial concentration of NTHi was
equivalent among the three cohorts as expected and ranged from
5.3×103 to 1.8×104 CFU NTHi/mg mucosa (FIG. 4).
Within three days after primary TCI, significantly fewer NTHi were
detected in mucosal homogenates from animals administered either dmLT
alone or chimV4+ dmLT (8.5×102 and 1.5×103 CFU/mg
mucosa, respectively), compared to saline (3.5×104 CFU;
p<0.05). Moreover, the latter cohorts exhibited a 10-fold reduction in
CFU NTHi relative to three days prior, influenced by administration of
dmLT, as the two cohorts that received this adjuvant were distinct from
the cohort administered saline, yet not significant from each other.
Furthermore, this result could not be attributed to development of
adaptive immune response, as only three days had passed since primary
immunization and no immunogen-specific antibody was yet detectable within
MEFs, as described below. Over the following four days, the cohort that
received dmLT alone maintained a bacterial concentration of
1.5-1.8×103 CFU NTHi/mg mucosa, however middle ears from
chinchillas immunized with chimV4+ dmLT exhibited a sequential 11- and
24-fold reduction in CFU on days 5 and 7 after primary TCI, respectively
(p<0.05 compared to animals administered saline on days 5 and 7;
p<0.05 compared to animals administered dmLT alone on day 7 only).
Moreover, within the latter 7 days of the study, only 10-30 CFU NTHi/mg
tissue were detected (p<0.05 compared to receipt of saline; p<0.05
compared to receipt of dmLT alone on day 14 only). Thus, while TCI with
dmLT alone appeared to stimulate an initial reduction in CFU NTHi within
mucosal biofilms in the middle ear, the combined influence of chimV4+
dmLT yielded consistently fewer NTHi at each subsequent time point.

[0140] To begin to examine the mechanisms for the protective efficacy
observed, the relative quantity of immunogen-specific IgG and IgA within
MEFs was examined by ELISA. chimV4-specific IgG within MEFs collected
from animals immunized with chimV4+ dmLT was detected at levels above
background beginning 5 days after primary TCI (GMT=80; Table 1). Whereas
a slight, but non-significant decrease in titer was observed on day 7,
receipt of the second immunizing dose induced a 2.5-fold increase in
specific IgG to a GMT of 160, which was maintained for an additional two
days (p<0.05 compared to day 0). By day 14, seven days after receipt
of the second immunizing dose, the GMT of chimV4-specific IgG in the
middle ear equaled 190 (p<0.05 compared to day 0). Whereas
chimV4-specific IgA was also detected within MEFs beginning five days
after primary TCI, the relative quantity of this antibody isotype was
2.0-4.6-fold less than IgG at each time point tested and no significant
increase in quantity of IgA was achieved relative to pre-immunization
values (Table 2). The ability to eradicate NTHi from the middle ear was
associated with a GMT value of at least 160 for IgG. Review of the
kinetics of NTHi clearance from the MEF of chinchillas immunized with
chimV4+ dmLT (FIG. 2) further supports this observation as on days 9-14,
time points at which the GMT of chimV4-specific IgG 160, significantly
fewer NTHi were present within MEFs and a >5-log decrease in CFU was
achieved.

[0141] Geometric mean of the reciprocal titer for chimV4-specific IgG and
IgA within MEFs collected from chinchillas immunized by TCI with chimV4+
dmLT. 95% confidence intervals of the geometric mean are indicated in
brackets. N=4 middle ears for day 0, N=6 middle ears for days 3 to 14.

[0142] As reduction of established NTHi mucosal biofilms began within
three days after primary TCI (FIG. 4) and prior to detection of antibody
in MEFs (Table 1), it was hypothesized that production of innate host
defense molecules by the middle ear epithelium contributed to this
observation. One of many such effectors, cBD-1, the orthologue to human
3-defensin3, known to be expressed in the chinchilla uppermost
respiratory tract that exhibits bactericidal activity against NTHi in
vitro and shown to have a role in the control of NTHi during experimental
colonization of the chinchilla nasopharynx. Three days after primary
immunization (seven days after NTHi challenge), 50.3±16.7 ng cBD-1/ml
was detected within homogenates of middle ear mucosa collected from
animals that received saline, compared to 643.4±57.3 ng/ml and
849.8±49.0 ng/ml in mucosae from animals administered dmLT only or
chimV4+ dmLT, respectively. These data show that the reduced bacterial
concentration detected within mucosal biofilms adherent to the middle ear
mucosa of animals immunized with dmLT alone and chimV4+ dmLT can be
attributed to significantly more cBD-1 present within this milieu,
compared to the cohort that received saline (p<0.05).

[0143] B (vi) Activation of Dermal DCs

[0144] As a means to examine the functional activation of dermal DCs after
TCI, pinnae were collected 3 h after immunization and cultured ex vivo.
At this arbitrarily selected time point, 1.5×103
CD11c+CD11b+DC-SIGN+ dermal DC/ml had emigrated from the
pinnae into the culture medium, a value which can be considered as
steady-state migration in this assay system. TCI with dmLT alone resulted
in 12-times more dermal DCs (1.9×104 cells/ml), although not a
significant result. The greatest and significant efflux of dermal DCs was
observed from pinnae on which chimV4+ dmLT was applied; a total of
6.8×104 cells/ml was observed, 44- and 4-fold more cells
compared to application of saline or dmLT alone, respectively
(p<0.01).

[0145] Activated DCs can secrete cytokines and chemokines as a means of
functional modulation via autocrine or paracrine manner. The supernatants
from the cultured pinnae were assayed for the presence of inflammatory
mediators via membrane array. Of a panel of 36 effectors, seven were
expressed at least 2-fold greater after TCI of pinnae with dmLT or
chimV4+ dmLT compared to administration of saline (FIG. 5). Of note is
the 10- and 5-fold increase in CCL4 induced by TCI with chimV4+ dmLT,
compared to saline or dmLT alone, respectively. While many of the listed
mediators are pluripotent and are involved in the induction of
inflammatory responses, a majority of these factors share a common role
as chemotactic agents for specific cell types. IL-23, IL-16, CCL5 and
CCL4 in particular, are noted to induce the migration and differentiation
of CD4+ T-cells, critical first steps toward the initiation of an immune
response.

[0146] B (vii) Induction of Polyfunctional CD4+ T-Cells

[0147] The phenotype of the resultant immune response, specifically, the
pattern of cytokine production by CD4+ T-cells within the NALT after TCI
with any of the three vaccine formulations, was examined. The NALT was
examined as the primary lymphoid aggregate to which dermal DCs trafficked
after TCI in this model. Compared to the NALT isolated from chinchillas
to which saline was applied, TCI with dmLT alone yielded 5.1% of CD4+
T-cells that produced both IFN-γ and IL-17 (FIG. 6), further
support for the induction of a modest response by administration of this
potent adjuvant alone. ChimV4 admixed with dmLT resulted in a 5.7-fold
increase in the percentage of T-cells that produced both IFN-γ and
IL-17--(29.3%), relative to receipt of dmLT alone. Moreover, within the
positive population highlighted, two subtypes were observed; one of
lesser dual fluorescence and one of greater dual fluorescence. Each
population may represent CD4+ T-cells of greater and lesser activation
states.

[0148] The greater percentage of CD4+ T-cells observed within the NALT of
animals immunized by TCI with chimV4+ dmLT, compared to receipt of dmLT
or saline alone, indicated that this immunization regime induced the
greatest expansion of CD4+ T-cells resident within this lymphoid
aggregate of the three formulations. Moreover, increase in the number of
cells that expressed both IFN-γ and IL-17, represented by the
greater overall fluorescent signal within this population, demonstrated
robust differentiation to cells that appeared functionally polarized
toward a dual Th1-Th17 phenotype. These polyfunctional T-cells have the
potential to further facilitate the development of a broadly protective
and multifactorial immune response that could be highly efficacious
against-induced experimental OM, an outcome which was observed herein.

[0149] C. Discussion

[0150] OM is a common disease of childhood and while the associated
mortality is very limited at least in developed countries, the morbidity
associated with this disease in terms of developmental delays, quality of
life and economic impact is substantial. The chronic nature of OM may be
attributed to the ability of the bacterial causative agents, including
NTHi, to form mucosal biofilms within the middle ear space. These highly
structured communities are recalcitrant to antibiotic therapies and
resist immune-mediated clearance. Among many other proteins, and an
abundance of host- and bacterial-derived extra-cellular DNA, NTHi OMP P5
and Tfp are identified as components of the biofilm matrix. TCI with
chimV4, a chimeric immunogen designed to target both of these critical
NTHi adhesions (outer membrane protein P5 and Tfp), delivered with dmLT
by simply rubbing the formulations on to the pinnae of chinchillas was
significantly efficacious to both prevent NTHi-induced OM and resolve
active OM. The rapidity with which biofilms that had been established
within the middle ear resolved was striking. TCI with NTHi
adhesin-directed immunogens plus dmLT induced the activation and
subsequent migration of cutaneous DCs to the NALT, which is located in
close proximity to the site of experimental disease, and thereby
facilitated the efficacy observed.

[0151] The mechanisms that afforded the observed efficacy and examine the
kinetics of disease resolution were examined utilizing an established
chinchilla model wherein cohorts of animals were first challenged by
direct inoculation of the middle ear with NTHi to induce OM. The
chinchilla serves as a relevant model for this pediatric disease as it is
permissive to infection with the viral and bacterial agents that cause OM
in humans and by appropriate viral-bacterial co-infection, one can
experimentally mimic the natural disease course of OM observed in the
child. It has shown reproducibility in numerous preclinical trials
wherein the efficacy of NTHi-targeted immunogens to prevent or
resolve-induced OM was assessed and is shown to be predictive of a
clinical trial outcome in examined in children. Moreover, the chinchilla
also serves as a robust host that both we and others employ to study
characteristics of bacterial biofilms associated with OM.

[0152] In the rigorous challenge model described herein, after a period of
four days to permit NTHi to establish robust biofilms within the middle
ear, animals were immunized via TCI. The administration of saline served
as a negative control and provided the ability to examine the immune
response elicited by application of dmLT alone. This adjuvant is a form
of E. coli heat-labile enterotoxin wherein two amino acid substitutions
inactivate a trypsin cleavage site and modifies a potential pepsin
cleavage site, thus rendering the molecule nontoxic, while adjuvant
properties are retained. DmLT promotes activation of various cell types,
including DCs, with a resultant mixed Th1/Th2-type immune response.

[0153] Compared to delivery of saline, three days after primary TCI with
dmLT alone, an initial reduction in clinically-relevant signs of OM, in
addition to a significant decrease in CFU NTHi within middle ear mucosal
biofilms was achieved. However, this was a transient outcome, as two days
later no differences were observed between the two cohorts. This early
response likely reflected nonspecific activation and function of dermal
DCs. However, without a sustained antigenic stimulus, this response
appeared temporary. In contrast, the formulation in which chimV4 was
administered with dmLT induced a similar, yet, prolonged and effective
outcome. Significant differences in clinically-relevant signs of OM,
significant reduction in the amount of mucosal biofilms within the middle
ear space and significantly fewer NTHi were present within the biofilms
beginning three days after receipt of the first immunizing dose. As
immunogen specific IgG and IgA was not detected within the middle ear
until 5 days after primary TCI, these early effects represent activation
of innate immune elements, i.e. dendritic cell activation, production of
host defense peptides, cytokine/chemokine signaling. The observed greater
efflux of dDCs from the pinnae and detection of a greater concentration
of cBD1 support this. Thus, our data shows that dmLT played an early,
critical role in immune activation that was further enhanced and focused
against NTHi with the addition of chimV4 in the vaccine formulation.

[0154] Host defense peptides are multifunctional proteins that possess
both antimicrobial and immunomodulatory characteristics. The 3-defensins
are one class of host defense peptide and are shown to be produced by
epithelial cells, constitutively or immediately in response to infection.
Middle ear mucosal homogenates were tested for the presence of cBD-1, the
orthologue to human 3-defensin 3, as a potential innate immune factor
that contributed to the early reduction in signs of OM and concentration
of NTHi within mucosal biofilms. A correlation between a greater
concentration of cBD-1 and fewer NTHi was detected, particularly within
the middle ears of animals that received dmLT alone or chimV4+ dmLT. It
is also reported that exposure of neutrophils to human 3-defensin-3
suppresses apoptosis of these critical immune cells; moreover this
molecule is chemotactic for neutrophils and macrophages. While these
functions are not yet described for cBD-1, this molecule may possess
similar functional characteristics, the result of which would be the
elimination of NTHi from the middle ear, as observed.

[0155] Specific to the kinetics of disease resolution, the first seven
days after primary TCI yielded the most dramatic resolution of disease,
shown by multiple assessments. Via video otoscopy and tympanometry,
delivery of chimV4+ dmLT resulted in a 90% reduction in the percentage of
middle ears with OM within this cohort. Grossly, by day 7 the mean
mucosal biofilm score for this cohort equaled 1.7, a 50-75% reduction
relative to day 0. Moreover, a >5-log reduction in CFU NTHi was
detected within mucosal biofilms collected from the middle ear. The only
assessment that did not follow this trend was the examination of CFU NTHi
within MEFs. No differences were detected among the cohorts until
beginning on day 7, at which time the planktonic NTHi were rapidly
eradicated. Interestingly, the time point for resolution of NTHi from
MEFs correlated with the detection of chimV4-specific IgG in MEFs at a
GMT of 160. As previously mentioned, a GMT of 160 is associated with
resolution of experimental OM in this model.

[0156] Collectively, our data demonstrated two phases of disease
resolution in this experimental model: an early phase from days 0 to 7
after primary TCI in which dmLT-induced activation of dermal DCs with
subsequent migration to the NALT, secretion of cytokines and chemokines
by activated DCs to stimulate expansion and polarization of CD4+
T-cells and production of at least one of many host defense peptides
known to have activity against NTHi, cBD-1. Inclusion of chimV4 within
the formulation prolonged this response.

[0157] Phase two began 7 days after primary TCI, upon administration of
the second immunizing dose as immunogen-specific antibody that targets
determinants expressed by NTHi was now detected. While the timeframe
proposed by this model incorporates the basic tenets for the initiation
of innate and adaptive immune responses, this is the first report of a
kinetic analysis of disease resolution after TCI that also incorporates
challenge with a biological agent.

Example 2

Synthesis of Chimeric Proteins

[0158] The chimeric proteins of the presently described subject matter
were produced using standard 15 recombinant methods. Initially, a
gene-synthesis company, (Blue Heron Biotechnology Inc.) was contracted to
make the initial plasmid based on the chimeric protein amino acid
sequences described herein that were optimized for E. coli preferred
codon usage. Briefly, the native NTHi pilin protein sequence was modified
by truncating the N-terminus (residues 1-39 of SEQ ID NO: 2) and adding a
HIS-tag sequence and a thrombin cleavage site as set out in SEQ ID NO: 3.
The HIS-tag was preceded by a sequence (MUSS) to assist in expression.
The thrombin cleavage site allowed for release of the HIS-tag. These
plasmids were then cloned into the E. coli expression vector pET-15b
vector (Novagen). The plasmid were then transformed into E. coli strain
"Origami(DE3)" (available from Novagen) as the host for expression of
soluble His-tagged chimeric proteins. Another E. coli host cell
expression stain that may be used is Origami B(DE3) (Novagen).

[0159] The His-tagged variants of the chimeric proteins will be recovered
by nickel column chromatography, then used for initial studies to
determine if they are reactive with antisera directed against any of the
following: native OMP P5-fimbrin, LB1 (full length 40 amino acid
peptide), LB1(1) (a synthetic peptide representing just the 19 amino acid
B-cell epitope of LB1), recombinant PilA protein or native PilA protein.
Once the His-tag is removed by thrombin site cleavage, the recombinant
huneric proteins will be used as immunogens to determine their
immunogenicity and protective capability.

[0160] Exemplary chimeric proteins of the presently described subject
matter have the sequences as set out in Table 2 below. The chimeric
proteins having the amino acid sequences of SEQ ID NOS: 10, 12 and 14
have been expressed by E. coli as described above.

[0161] Additional exemplary chimeric proteins of the presently described
subject matter have the amino acid sequences as set forth in Table 3
below. These chimeric proteins have been expressed by E. coli and
purified using a HIS-tag, as described above. The chimeric proteins set
out in Table 3 have the His tag sequence removed for use as an immunogen.
The chimeric protein having the amino acid sequence of SEQ ID NO: 56 was
used in the studies described in Example 5.

[0162] All publications cited in the specification are indicative of the
level of skill of those skilled in the art to which the presently
described subject matter pertains. All of these publications are hereby
incorporated by reference herein to the same extent as if each individual
publication were specifically and individually indicated as being
incorporated by reference.

[0163] The present subject matter being thus described, it will be
apparent that the same may be modified or varied in many ways. Such
modifications and variations are not to be regarded as a departure from
the spirit and scope of the present subject matter, and all such
modifications and variations are intended to be included within the scope
of the following claims.